The ubiquitous Hsp70 family of molecular chaperones utilizes a deceptively simple mechanism of interdomain allostery to accomplish a stunningly broad array of critical cellular functions. Hsp70s facilitate folding of newly synthesizd proteins, protect cells from damage that can occur under stress conditions by either rescuing misfolded proteins or directing them to degradation machinery, assist protein translocation across membranes, and promote assembly and disassembly of macromolecular complexes. All of these functions rely on the ability of Hsp70s to bind unfolded regions of a protein substrate, and to release their substrates upon allosteric binding of ATP. Hsp70s have emerged as potential targets for cancer therapeutics because of their anti-apoptotic and tumorigenic activity, and as targets for diseases associated with protein misfolding (cystic fibrosis, neurodegenerative diseases) because of their ability to rescue aggregation-prone proteins. Our work using the E. coli Hsp70, DnaK, as a paradigm for the fundamental mechanism of Hsp70 allostery has shown how allosteric signals are communicated from the nucleotide-binding site of the N-terminal domain to the interdomain linker, and we have tantalizing hints about how this then causes major reorganization and concomitant reduced substrate affinity in the C-terminal substrate-binding domain (SBD). In Aim 1, we will build on our recent progress and map the allosteric signal transmission in DnaK into the SBD, using a combination of novel nuclear magnetic resonance approaches for large proteins, and complementary biophysical methods such as pulsed electron spin resonance. We will study how co-chaperone interactions remodel the DnaK allosteric landscape, and how our newly discovered role for the extreme C-terminus of DnaK may influence its interactions with substrates. In Aim 2, the methods and knowledge gathered through our ongoing work on DnaK will be applied to four human Hsp70s, the cytoplasmic Hsc70 (constitutive) and HspA1 (inducible), the ER-resident BiP, and the mitochondrial mtHsp70. These chaperone machines share a common mechanism but are characterized by substantial sequence diversification and consequent differences in substrate preferences and in co-chaperone partners. In Aim 3, we will structurally and biophysically characterize small molecule modulators of Hsp70s provided to us by collaborators who are exploring Hsp70s as drug targets. We will determine by NMR the mode of interaction of candidate small molecules with Hsp70s, look for evidence of selectivity for particular Hsp70s and how it may be enhanced, and provide feedback to improve characteristics of modulators. Overall, the research proposed will contribute to our fundamental understanding of allostery, while providing insights into function and specialization of different Hsp70 family members. These insights are critical for optimal use of Hsp70s as drug targets.